Many agriculturally important crops are susceptible to infection by plant viruses. These viruses can seriously damage a crop and drastically reduce its economic value to the grower. This eventually leads to a higher cost for the consumer. Attempts to control or prevent infection of a crop by a plant virus have been made, yet viral pathogens continue to be a significant problem in agriculture.
Scientists have recently developed means to produce virus resistant plants using genetic engineering techniques. Such an approach is advantageous in that the means for providing the protection is incorporated in the plant itself and can be passed on to its progeny. A host plant is resistant if it possesses the ability to suppress or retard the multiplication of a virus, or the development of pathogenic symptoms. "Resistant" is the opposite of "susceptible", and may be divided into: (1) high, (2) moderate, or (3) low resistance, depending upon its effectiveness. Essentially, a resistant plant shows reduced or no symptom expression, and virus multiplication within it is reduced or negligible. Several different types of host resistance to viruses are recognized. The host may be resistant to: (1) establishment of infection, (2) virus multiplication, or (3) viral movement.
It has previously been shown that expression of a plant virus capsid protein, which is termed the coat protein, in a plant can confer resistance to the homologous virus and to related viruses (Abel et al. 1986; Tumer et al. 1987: Cuozzo et al. 1988; Hemenway et al. 1988; Stark and Beachy 1989; Lawson et al. 1990; Kaniewski et al. 1990). In these studies, resistance to virus disease is defined as either reduced incidence of infection, delayed symptom development, reduced virus replication or viral antigen levels, or slower to no systemic virus movement. Expression of the virus coat protein in these transgenic plants is responsible for the observed effects in the reduction of virus disease by an as yet undetermined mechanism (Abel et al. 1986; van Dun et al. 1988). This type of protection against viral infection is termed coat proteinmediated protection.
Even though coat protein-mediated viral resistance has proven to be useful in variety of situations, it may not always be the most effective or the most desirable means for providing viral resistance. In such instances, it would be advantageous to have other methods for conferring viral resistance to plants.
A fragment of the putative replicase gene from tobacco mosaic virus (TMV) recently has been found to provide resistance against TMV when expressed in tobacco plants (Golemboski et al. 1990). In TMV, two proteins, the 183 kDa and 126 kDa proteins, have been speculated to be replicase components, as the expression of both proteins are necessary for normal multiplication in tobacco plants (Ishikawa et al. 1986). The 183 kDa protein is a readthrough product of the 126 kDa sequence. The 126 kDa protein contains the NTP binding motif. The 183 kDa protein contains both the NTP and GDD motifs. More specifically, the 54 kDa readthrough portion of the 183 kDa protein is the portion that contains the GDD motif. Golemboski et al. (1990) found that transgenic tobacco plants expressing the 54 kDa read-through portion were protected against infection by TMV. They did not, however, observe protection in transgenic plants expressing the larger 126 kDa protein. Moreover, they did not report any protection data from experiments in which the plants expressed the 183 kDa protein.
Others have conducted protection experiments with transgenic plants expressing components of non-structural viral proteins. For example, van Dun et al. (1988) analyzed protection in tobacco plants expressing either of two genes encoding proteins involved in the replication of alfalfa mosaic virus (AlMV). These plants were transformed with RNAs 1 or 2 of AlMV, which encode proteins P1 and P2, respectively. The polypeptides encoded by these RNAs have amino acid homologies to other vital replicases, and both RNAs are known to be essential for replication. In contrast to the PVY ORF1, the NTP and GDD binding motifs for AlMV reside on different RNAs and consequently different proteins. The GDD domain contains a glycine amino acid residue (G) followed by two aspartate amino acid residues (D). P1 on RNA1 has homology to the NTP binding motif and P2 on RNA2 has homology to the GDD motif. Plants expressing either RNA1 or RNA2 were not protected against infection by AlMV. In addition, plants expressing both RNAs 1 and 2 were likewise not protected against infection by AlMV (Taschner et al. 1991).
Buck et al. (PCT publication WO 92/03539) have described the use of various techniques to prevent the expression or function of a cucumber mosaic viral replicase in order to provide viral resistance in plants, including the expression of a fragment of the replicase gene in order to provide this viral resistance. The techniques employed or disclosed in this publication to accomplish this included: (1) antisense technology (wherein a complementary RNA to that coding for the full length replicase was expressed); (2) expression of a gene coding for an antibody specific for one of the three components of the replicase (viral encoded polypeptides P1a and P2a, and polypeptide P50 from tobacco); (3) a truncated form or fragment of the replicase; and (4) use of a ribozyme specific for the RNA coding for one of the components of the replicase.
Potato virus Y (hereinafter PVY) is a member of the potyvirus plant virus group. The potyvirus group of plant viruses comprises the largest group of plant viruses which flourish in a wide range of crops and environmental conditions. Representative members of the potyvirus group include, but are not limited to, potato virus Y (PVY), tobacco vein mottling virus, watermelon mosaic virus, zucchini yellow mosaic virus, bean common mosaic virus, bean yellow mosaic virus, soybean mosaic virus, peanut mottle virus, beet mosaic virus, wheat streak mosaic virus, maize dwarf mosaic virus, sorghum mosaic virus, sugarcane mosaic virus, johnsongrass mosaic virus, plum pox virus, tobacco etch virus, sweet potato feathery mottle virus, yam mosaic virus, and papaya ringspot virus. PVY is a positive-sense, single-stranded RNA virus that is surrounded by a repeating proteinaceous monomer, which is termed the coat protein (CP). The encapsidated virus has a flexous rod morphology, which is characteristic of the potyvirus group. The majority of the potyviruses are transmitted in a nonpersistent manner by aphids. A list of potyviruses causing serious annual crop losses and the crop species affected are shown in Table 1. As can be seen from the wide range of crops affected by potyviruses, the host range includes such diverse families of plants, but is not limited to Solanaceae, Chenopodiaceae, Gramineae, Compositae, Leguminosae, Dioscoreaceae, Cucurbitaceae, and Caricaceae. The various potyviruses also demonstrate cross-infectivity between plant members of the different families.
TABLE 1 ______________________________________ Representative Potyviruses causing serious annual crop losses and the crop species affected: ______________________________________ Potato virus Y potato, tobacco, tomato, pepper Tobacco etch tomato, pepper Watermelon mosaic cucurbits Zucchini yellow mosaic cucurbits Bean common mosaic Phaseolus sp. Bean yellow mosaic Phaseolus sp. Soybean mosaic soybeans Peanut mottle peanuts, beans, soybeans, peas Beet mosaic sugarbeets, spinach Wheat streak mosaic small grains, e.g. wheat Maize dwarf mosaic corn, sugarcane, sorghum Sorghum mosaic corn, sugarcane, sorghum Johnsongrass mosaic corn, sugarcane, sorghum Plum pox plum, peach, nectarine, apricot Papaya ringspot papaya Tobacco vein mottling tobacco Sugar cane mosaic sugarcane yam mosaic yam sweet potato feathery mottle sweet potato ______________________________________
The host range of PVY is mainly restricted to members of the Solanaceae family, including potato, tobacco, and tomato (Purcifull and Edwardson, 1981). Potato plants are particularly susceptible to infection by PVY. Infection by PVY of a potato crop results in a substantially reduced yield. Moreover, simultaneous infection by PVY and the potexvirus Potato virus X (PVX) causes a devastating synergistic effect. A combined PVY and PVX infection can reduce yields as much as 90% (deBokx, 1986; Vance 1991). Other synergistic infectious relationships between a potyvirus and another type of plant virus are also known to exist. Further examples of this synergism include the following: (1) the combined infection of maize dwarf mosaic virus ( a potyvirus) and maize chlorotic mottle virus in corn to produce a disease known as corn lethal necrosis; and (2) the combined infection of potato virus Y (a potyvirus) and cucumber mosaic virus in tomato. This synergistic relationship between the viruses during the course of infection is in all cases marked by a dramatic increase in the total damage generated in the plant.
PVY is an aphid-transmitted virus. Because of the ease of transmission of the virus by aphids, potato growers must adhere to strict cultural practices to control PVY infection. These methods include isolating plants from sources of infection, wide spacing to avoid plant contact, and the use of insecticides to control aphid populations. Additionally, fields must be visually inspected for morphological signs of infection; plants showing visible signs of PVY infection are destroyed by the grower. Because most of the commercial potato cultivars are propagated vegetatively, maintaining virus-free lines is of critical importance to the potato grower. Farmers plant `potato seeds`, which are not actually true seeds, but rather pieces of potato tubers. Before `potato seeds` can be sold to the farmer, they must pass a rigorous series of tests to be certified virus free. PVY infection of `potato seeds` is a cause of seed decertification. Commercially important potato cultivars, to which the present invention may be applied, include but are not limited to Russet Burbank, Shepody, Atlantic, Norchip, and Superior.
The PVY genomic RNA replicates through RNA intermediates in a DNA-independent fashion. PVY RNA has one open reading frame (ORF) that codes for a polyprotein of 352 kilodaltons (kDa). This polyprotein undergoes proteolytic processing by viral encoded proteases and a host protease to yield around or about eight viral proteins of 31 kDa, 62 kDa, 38 kDa, 71 kDa, 5.6 kDa, 50 kDa, 60 kDa, and 30 kDa. These proteins can be found in the infected plant cell and form the necessary components for viral replication. The 50 kDa viral encoded protein known as the nuclear inclusion I or nuclear inclusion A protein (hereinafter referred to as protease) contains the proteolytic activity responsible for the cleavage of the carboxy 2/3 of the viral polyprotein to yield the 71 kDa, 5.6 kDa, 50 kDa, 60 kDa, and 30 kDa proteins. Additionally, the protease contains a domain which covalently links to the 5' terminus of the viral genomic RNA. This domain, or viral protein genome linked (VpG), presumably functions in replication of the viral genomic RNA. In the course of a potyviral infection, the protease (50 kDa) and the 60 kDa protein (also referred to as the nuclear inclusion II or nuclear inclusion B protein or replicase) are transported into the nucleus of the plant cell and accumulate to high levels.
In general, a protease gene derived from a particular potyvirus shares certain common features with protease genes derived from other potyviruses. One of these common features is the location of the gene within the potyviral genome. The protease gene is typically located directly upstream from the potyviral replicase or nuclear inclusion II gene, which is itself located directly upstream from the potyviral coat protein. A second common feature is that there is a defined type of cleavage site with a consensus sequence, which is self-processed by the potyviral protease during cleavage of the polyprotein. This protease or nuclear inclusion A or I is responsible for the proteolytic processing of the C--terminal 2/3 of the polyprotein. Yet a third common feature is the presence of the VpG domain in potyviral proteases, which was discussed earlier herein. A fourth common feature is the relatedness of the overall sequence of the potyviral protease with other known protease genes. In particular, the potyviral protease protein contains amino acids that are characteristic of other serine proteases. [See Riechmann et al., Journal of General Virology, (1992) 73:1-16; and Garcia et al., Virology, (1992) 188:697-703].
In the course of a potyviral infection, the protease (50 kDa) protein and the replicase protein (60 kDa, also referred to as the nuclear inclusion II or nuclear inclusion B protein) are posttranslationally transported across the nuclear membrane into the nucleus of the plant cell at the later stages of vital infection and accumulate to high levels. Generally speaking, transport across the nuclear envelope is an active process mediated by a nuclear localization signal (NLS) contained within the primary sequence of the transported protein. Unlike classical signal sequences, which are generally located at the N-terminus, nuclear localization signals may be found at any site within the protein, including the N-terminus. Nuclear localization signals have been identified as sequences that may by genetic or biochemical fusion render a cytoplasmic protein nuclear, or when deleted or mutated, may no longer promote nuclear uptake of the protein in which they reside.
NLSs are typically short sequences (8-10 amino acids), contain a high proportion of positively charged amino acids (lysine and arginine), are not located at specific sites within the protein, are not removed following localization, and can occur at more than one site within the protein. Nuclear localization signals may interact with the nuclear pore complex or, possibly, cytoplasmic components. [See Garcia-Bustos et al. (1991) for a recent discussion of nuclear protein localization]. Here, the accumulation of the potyvirus protease in the nucleus at later stages of infection indicates that the presence of protease in the cytoplasm of the cell at that phase may actually interfere with some aspect of viral assembly. Although not fully understood, the over-accumulation of the potyvirus protease in the cytoplasm of the plant cell by the over-expression of the protein, or the prevention of nuclear localization at a particular stage in the viral replication may be the mechanism by which resistance to the potyvirus is conferred to the transformed plant. Alternate means by which this resistance could be conferred include the binding of the viral RNA by the protease and subsequent transport of the complex to the nucleus; binding of the viral RNA and prevention of its assemby into viral particles; or a disturbance of the balance between various required components and subsequent interference with the assembly of an active enzyme complex.
As can be seen from the foregoing discussion, potyviral infection of various plants is a serious problem encountered in agriculture today. Thus, it would be a significant contribution to the art to develop an alternative method to those currently available that is effective for conferring viral resistance to plants,